Ultrasensitive dopamine detection with graphene aptasensor multitransistor arrays

Detecting physiological levels of neurotransmitters in biological samples can advance our understanding of brain disorders and lead to improved diagnostics and therapeutics. However, neurotransmitter sensors for real-world applications must reliably detect low concentrations of target analytes from small volume working samples. Herein, a platform for robust and ultrasensitive detection of dopamine, an essential neurotransmitter that underlies several brain disorders, based on graphene multitransistor arrays (gMTAs) functionalized with a selective DNA aptamer is presented. High-yield scalable methodologies optimized at the wafer level were employed to integrate multiple graphene transistors on small-size chips (4.5 × 4.5 mm). The multiple sensor array configuration permits independent and simultaneous replicate measurements of the same sample that produce robust average data, reducing sources of measurement variability. This procedure allowed sensitive and reproducible dopamine detection in ultra-low concentrations from small volume samples across physiological buffers and high ionic strength complex biological samples. The obtained limit-of-detection was 1 aM (10–18) with dynamic detection ranges spanning 10 orders of magnitude up to 100 µM (10–8), and a 22 mV/decade peak sensitivity in artificial cerebral spinal fluid. Dopamine detection in dopamine-depleted brain homogenates spiked with dopamine was also possible with a LOD of 1 aM, overcoming sensitivity losses typically observed in ion-sensitive sensors in complex biological samples. Furthermore, we show that our gMTAs platform can detect minimal changes in dopamine concentrations in small working volume samples (2 µL) of cerebral spinal fluid samples obtained from a mouse model of Parkinson’s Disease. The platform presented in this work can lead the way to graphene-based neurotransmitter sensors suitable for real-world academic and pre-clinical pharmaceutical research as well as clinical diagnosis. Supplementary Information The online version contains supplementary material available at 10.1186/s12951-022-01695-0.

. Raman spectrum of a CVD-grown graphene sample transferred onto a Si/SiO2 substrate.  Figure S2. Individual transistor (black circles) and averaged (red lines) responses (ΔVDirac) from a single gMTA chip to a blank sample and to two samples with different concentrations of the analyte.

Figure S3: XPS peak fitting for PBASE and PBASE + aptamer samples
To confirm and further analyze the PBASE + aptamer bond for the implemented biofunctionalization process, high-resolution XPS spectra of C 1s, O 1s, and N 1s were fitted with Avantage data processing software (Thermo Fisher Scientific). Smart-type background subtraction was used for peak fitting, and quantification was done using sensitivity factors provided by the Avantage library. The XPS results support the successful binding of the DNA aptamer to PBASE in the substrate. Considering that the DNA aptamer binds to either the carbonyl group (C=O) or the carboxylate (C-O) part of the crosslinker (3), a reduction of these peaks, at approximately 287 eV and 285.5 eV, respectively, was observed in the PBASE + aptamer sample when compared with the PBASE sample ( Fig. S2-A). The C-O bond peak from the PBASE sample became a C-N bond peak at approximately 286 eV in the PBASE + aptamer due to new contributions of C-O-C and C-OH bonds from the DNA aptamer sugar unit ( Fig. S2-A) (4). A decrease of the N-(C=O)-O-bond peak at approximately 401.5 eV and an increase of the aromatic peak at approximately 399.4 eV was observed in the PBASE + aptamer sample when compared with the PBASE sample due to the amine termination of the DNA aptamer strand (Fig. S2-B). Two peaks were observed in the O 1s spectrum, a C=O bond peak at approximately 533 eV, which is from PBASE's aromatic ring, and an H-C-O bond peak at approximately 535 eV, which is from the PBASE's strand that links directly to the amino group from DNA ( Fig. S2-C) (4). The H-C-O bond observed in the PBASE sample was not present in the PBASE + aptamer sample, likely due to the DNA aptamer amino group's binding to the crosslinker. For the same reason, the C=O bond peak in the PBASE + aptamer sample increases compared with the PBASE sample ( Fig. S2-A).

Figure S4: Blank samples measurements in PBS
To correctly determine the limit-of-blank of our gMTAs to offset the calibration curves, 20 µL samples not containing dopamine molecules but prepared from dopamine stock solution diluted to zM (10 -21 ) in 1 × PBS, were incubated for 20 minutes. Figure S3 shows the observed transconductance shifts from baseline (ΔVDIRAC) for the blank samples, with an average of 19.9 ± 1.3 mV. Figure S4. Transconductance shifts from baseline (ΔVDIRAC) for blank samples not containing dopamine in 1 × PBS (left). Data is mean ± sem.

Figure S5: Stability of gMTA measurements
To assess the stability of our measurements, 20 µL 1X PBS samples were continuously incubated in one gMTA for 3 hours, and transconductance measurements were taken every 60 min. The coefficient of variation (CV) was calculated as the standard deviation to the measurements' mean ratio. A low CV of 1.13% was obtained, which indicates high stability. Figure S5. Transconductance (VDIRAC) measurements of PBS samples over time. Data is mean ± sem.

Figure S6: Dopamine attomolar detection with short incubation time
To assess if the gMTAs could detect dopamine in ultra-low concentrations with short incubations times, dopamine samples in 1x PBS from 0.1 aM (10 -19 ) to 100 aM (10 -16 ) were incubated for 5 minutes and the responses compared with those obtained for 1 hour incubation. Figure S5 shows that transconductance measurements after 5 min sample incubation are comparable to those obtained for 1 hour sample incubation and that an LOD of 1 aM still present. Figure S6. Calibration curves for dopamine detection in 1 × PBS with sample incubation times of 1h (red) and 5 min (brown) (data is mean ± sem, with 2 nd order polynomial line fit).